Sharlene G. Rakoczy

Catalase expression in delayed and premature aging mouse models.

The physiological decline that occurs with aging is thought to result, in part, from accumulation of oxidative damage produced by reactive oxygen species (ROS) generated during normal metabolism. Two genetic mouse models of aging, the Ames dwarf and growth hormone (GH) transgenic, suggest that hormone levels may play a role in antioxidative defense and aging. To explore this possibility, catalase (CAT), an enzyme involved in elimination of ROS, was evaluated in long-lived dwarf and short-lived transgenic mice. Catalase activity and/or protein was significantly elevated in livers from dwarf mice at 3, 6, 13-15, and 24 months of age when compared to age-matched wild type mice. In contrast, a 50 and 38% reduction (P<0.05) in CAT protein was observed in 3 and 10 to 12 month old GH transgenics respectively, when compared to wild type mice. Kidneys from old dwarf mice exhibited significantly increased CAT activity (22%), protein (16%) and mRNA expression (59%) compared to wild type mice. Conversely, kidneys from GH transgenic mice showed reductions in CAT activity. The results of this study suggest that hormonal status modulates antioxidative mechanisms and that CAT is important in overall defense capacity with respect to lifespan in both decelerated (dwarf) and accelerated (transgenic) mammalian models of aging.

Effects of Growth Hormone and Insulin-Like Growth Factor-1 on Hepatocyte Antioxidative Enzymes

The physiological decline that occurs in aging is thought to result, in part, from accumulation of oxidative damage generated by reactive oxygen species during normal metabolic processes. Elevated levels of antioxidative enzymes in liver tissues are present in the Ames dwarf, a growth hormone (GH)-deficient mouse that lives more than 1 year longer than wild-type mice from the same line. In contrast, transgenic mice that overexpress GH exhibit depressed hepatic levels of catalase and have significantly shortened life spans. In this study, we evaluated the in vitro effects of GH and Insulin-like growth factor 1 (IGF-1) on antioxidative enzymes in mouse hepatocytes. Hepatocytes were isolated from wild-type mice following perfusion of livers with a collagenase-based buffer. Dispersed cells were plated on Matrlgel and treated with rat GH (0.1, 1.0, or 10 μg/ml) or IGF-1 (0.5, 5.0, or 50 nM) for 24 hr. Hepatocytes were recovered and protein was extracted for immunoblotting and enzyme activity assays of catalase (CAT), glutathione peroxidase (GPX), and manganese superoxide dismutase (MnSOD). A 41% and 27% decrease in catalase activity was detected in cells treated with GH, whereas IGF-1 reduced CAT activity levels to a greater extent than GH (P < 0.0001). The activity and protein levels of GPX were also significantly depressed In cells treated with GH, whereas activity alone was decreased in cells treated with IGF-1 (P < 0.04). GH significantly suppressed MnSOD levels by 40% and 66% in 1.0 and 0.1 μg/ml concentrations, respectively. Similarly, IGF-1 decreased MnSOD protein levels (5 nM; P < 0.05). These results suggest that GH and IGF-1 may decrease the ability of hepatocytes to counter oxidative stress. In addition, these experiments provide an explanation for the differing antioxidative defense capacity of GH-deficient versus GH-overexpressing mice, and they suggest that GH is directly involved in antioxidant regulation and the aging process.

Growth hormone administration to long-living dwarf mice alters multiple components of the antioxidative defense system.

Endocrine hormones are thought to be involved in processes that contribute to aging. Long-living dwarf mice are growth hormone (GH)-deficient and exhibit enhanced expression of antioxidative defense molecules when compared to normal, wild type littermates. In this study, 3- and 12-month-old Ames dwarf mice received with 50 microg GH or saline for 7 days. Tissues were collected and assayed for several antioxidant molecules. In addition to increased body and liver weights, GH treatment of dwarf mice decreased liver, kidney and heart catalase protein (P < 0.05). Catalase activity was significantly decreased in kidney and heart tissues of mice receiving GH compared to dwarf mice treated with saline. Glutathione peroxidase (GPX) protein was significantly reduced in liver, kidney and muscle of GH-treated mice (P < or = 0.03). Likewise, the activity of GPX was decreased in liver and kidney tissues following GH administration (P< or = 0.04). Exogenous GH increased glutathione levels in brain, muscle and liver (P< or = 0.03) compared to saline controls. This evidence, along with previous data, suggests that GH suppresses key components of systems that counter oxidative stress. Reductions in GH and IGF-1 signaling contribute to extended life spans in a variety of species, which may be partially explained by an increased ability to neutralize deleterious byproducts of metabolism.

Growth hormone alters methionine and glutathione metabolism in Ames dwarf mice.

Reduced signaling of the growth hormone (GH)/insulin-like growth factor-1(IGF-1)/insulin pathway is associated with extended life span in several species. Ames dwarf mice are GH and IGF-1 deficient and live 50-64% longer than wild type littermates (males and females, respectively). Previously, we have shown that Ames mice exhibit elevated levels of antioxidative enzymes and lower oxidative damage. To further explore the relationship between GH and antioxidant expression, we administered GH or saline to dwarf mice and evaluated components of the methionine and glutathione (GSH) metabolic pathways. Treatment of dwarf mice with GH significantly suppressed methionine adenosyltransferase (40 and 38%) and glycine-N-methyltransferase (44 and 43%) activities (in 3- and 12-month-old mice, respectively). Growth hormone treatment elevated kidney gamma-glutamyl-cysteine synthetase protein levels in 3- and 12-month-old dwarf mice. In contrast, the activity of the GSH degradation enzyme, gamma-glutamyl transpeptidase, was suppressed by GH administration in heart and liver. The activity of glutathione-S-transferase, an enzyme involved in detoxification, was also affected by GH treatment. Taken together, the current results along with data from previous studies support a role for growth hormone in the regulation of antioxidative defense and ultimately, life span in organisms with altered GH or IGF-1 signaling.

Glutathione metabolism in long-living Ames dwarf mice

Ames dwarf mice live significantly longer than their wild type siblings and exhibit elevated antioxidative defenses and reduced oxidative damage. This study was conducted to determine the levels of components of glutathione (GSH) synthesis, degradation and utilization in dwarf and wild type mice. Glutamate-cysteine ligase protein levels were significantly elevated in dwarf liver at 3 and 24 months of age and muscle tissue at all ages examined. In kidney, activity of gamma-glutamyltranspeptidase (GGT) was decreased 42, 30 and 33% in 3, 12 and 24-month-old dwarf mice compared to wild type mice (P<0.0001). In contrast, GSH-S-transferase (GST) activity was markedly elevated (85, 113 and 53%) in kidneys of 3, 12 and 24-month-old dwarf mice (P<0.0001). GGT activity was higher in hearts of young dwarf and wild type mice while GST activity tended to be greater in dwarf mice. Similar to liver and kidney, brain GGT activity was also lower in dwarf mice (P<0.0001). Results of these experiments coupled with previous data provide a mechanism to partially explain the enhanced resistance to oxidative insult and conceivably, the extended longevity of dwarf mice.

Mitochondrial oxidant generation and oxidative damage in Ames dwarf and GH transgenic mice

Aging is associated with an accumulation of oxidative damage to proteins, lipids and DNA. Cellular mechanisms designed to prevent oxidative damage decline with aging and in diseases associated with aging. A long-lived mouse, the Ames dwarf, exhibits growth hormone deficiency and heightened antioxidative defenses. In contrast, animals that over express GH have suppressed antioxidative capacity and live half as long as wild type mice. In this study, we examined the generation of H2O2 from liver mitochondria of Ames dwarf and wild type mice and determined the level of oxidative damage to proteins, lipids and DNA in various tissues of these animals. Dwarf liver mitochondria (24 months) produced less H2O2 than normal liver in the presence of succinate (p<0.03) and ADP (p<0.003). Levels of oxidative DNA damage (8ÕHdG) were variable and dependent on tissue and age in dwarf and normal mice. Forty-seven percent fewer protein carbonyls were detected in 24-month old dwarf liver tissue compared to controls (p<0.04). Forty percent more (p<0.04) protein carbonyls were detected in liver tissue (3-month old) of GH transgenic mice compared to wild types while 12 month old brain tissue had 53% more protein carbonyls compared to controls (p<0.005). Levels of liver malonaldehyde (lipid peroxidation) were not different at 3 and 12 months of age but were greater in Ames dwarf mice at 24 months compared to normal mice. Previous studies indicate a strong negative correlation between plasma GH levels and antioxidative defense. Taken together, these studies show that altered GH-signaling may contribute to differences in the generation of reactive oxygen species, the ability to counter oxidative stress and life span.

Long-living growth hormone receptor knockout mice: Potential mechanisms of altered stress resistance

Endocrine mutant mice have proven invaluable toward the quest to uncover mechanisms underlying longevity. Growth hormone (GH) and insulin-like growth factor (IGF) have been shown to be key players in physiological systems that contribute to aging processes including glucose metabolism, body composition and cellular protection. Examination of these mutant mice across several laboratories has revealed that differences exist in both the direction and magnitude of change, differences that may result in variation in life span. Growth hormone receptor knockout mice lack a functional GH receptor, therefore GH signaling is absent. These mice have been shown to lack the heightened oxidative defense mechanisms observed in other GH mutants yet live significantly longer than wild type mice. In this study, glutathione (GSH) and methionine (MET) metabolism was examined to determine the extent of variation in this mutant in comparison to the Ames dwarf, a mouse that exhibits delayed aging and life span extension of nearly 70%. Components of GSH and MET were altered in GHR KO compared to wild type controls. The results of these experiments suggest that these pathways may be partially responsible for differences observed in stress resistance and the capacity to respond to stressors, that in the long term, affect health and life span.

Long-lived Ames dwarf mouse exhibits increased antioxidant defense in skeletal muscle

Resting and exercised (both acute and chronic) hindlimb skeletal muscle from long-lived Ames dwarf and wild type mice at 3, 12, 18, and 24 months of age was tested for antioxidant enzyme activity and protein, non-enzymatic antioxidant ratios, mitochondrial hydrogen peroxide concentration, and plasma lactate levels. Differences were observed in GPX enzyme activity between mouse genotypes at all physical activity levels, with dwarf mice exhibiting depressed levels at younger ages (3 months: P = 0.09 [non-swim], P = 0.03 [acute swim], P = 0.04 [chronic swim]) and comparatively higher levels than wild type mice at older ages (18-24 months: P = 0.05 [acute swim], P = 0.07 [chronic swim]). Catalase enzyme activity and the GSH system rarely demonstrated significant differences between genotypes, regardless of age or activity. However, the chronic exercise group displayed a difference in GSH:GSSG ratios between mouse genotypes (P = 0.005). Plasma lactate concentrations were elevated in the wild type mice compared to the dwarf mice at all ages in all activity groups. These results suggest there are biological differences with regard to antioxidant defense that favor the Ames dwarf mouse in active and resting skeletal muscle when compared to wild type mice.

Long-living Ames dwarf mouse hepatocytes readily undergo apoptosis

Ames dwarf mice live 50-64% longer and exhibit upregulated antioxidative defenses and lower cellular damage when compared to age-matched wild-type littermates. Due to the relationship between aging and apoptosis, the purpose of this study was to compare basal levels of apoptosis-related proteins in dwarf and wild-type tissues and to compare the response of dwarf and wild-type primary hepatocytes to oxidative stress. Hepatocytes from dwarf and wild-type mice (6 month-old) were isolated using collagenase perfusion and treated with hydrogen peroxide. Viability, activity, protein levels, and morphological changes were evaluated. Procaspase-3 protein levels were increased in dwarf kidney and liver (p<0.05) while Bcl-2 protein levels were significantly higher in dwarf liver at 24 months of age. Bax protein levels were markedly elevated in several tissues at different ages and Bcl-2/Bax ratios were lower in many dwarf tissues. In culture, peroxide-treated dwarf hepatocytes showed lower viability (p<0.03) and higher caspase-3 activity induction when compared to peroxide-treated wild-type cells. Peroxide-treated dwarf hepatocytes frequently showed morphological characteristics reminiscent of apoptosis, which were not observed in peroxide-treated wild-type hepatocytes. This suggests that when experiencing an oxidative challenge, Ames dwarf hepatocytes more readily undergo apoptosis than wild-type cells, providing an advantage to dwarf mice, whereby they more efficiently eliminate damaged cells, thus contributing to their longer lives.

Increases in insulin-like growth factor-1 level and peroxidative damage after gestational ethanol exposure in rats

Ethanol exposure during pregnancy elicits profound detrimental developmental and behavioral effects such as reduced levels of insulin-like growth factor-1 (IGF-1) in the fetus. However, few reports have addressed its impact on postpartum dams. This study was designed to examine the influence of gestational ethanol exposure on postpartum maternal organ oxidative damage and IGF-1 level. Pregnant female rats were pair-fed from Day 2 of gestation until labor with control or ethanol (6.36% (v/v)) liquid diets and were sacrificed 6 weeks after parturition (ethanol withdrawn after parturition). There was no difference in body weight during or after the gestational period between the control and ethanol groups. Litter size was significantly less for ethanol-fed dams. One-week postnatal pup survival was significantly lower in the ethanol-fed (57.1%) than the control (97.8%) group. Liver and kidney tissue IGF-1 levels and mRNA were elevated in the ethanol-fed mothers, accompanied by hepatic but not renal oxidative damage, indicated by profound lipid peroxidation (measured by malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE)) and protein carbonyl formation. The levels of glutathione (GSH), glutathione disulfide (GSSG) and GSH/GSSG ratios in liver and kidney were not different between the ethanol-fed and control dams. Collectively, these data suggest that gestational ethanol exposure may lead to postpartum oxidative organ damage and a possible compensatory increase in organ IGF-1 levels.